The reversible nature of the protonation and deprotonation sequence among the conjugated dienes implicated in the acid-catalysed double bond migration of steroid 5,7-dienes to 8,14-dienes allowed a high incorporation of deuterium atoms into the 5α-steroid 8,14-dienes. Treatment of cholesta-5,7-dien-3β-ol with a 37% deuterium oxide solution of deuterium chloride in refluxing O-deuterioethanol for 5 h afforded [5,6,6,7,7,11,11,12,12,15,16,16,17-2Hn]-5α-cholesta-8,14-dien-3β-ol 3a in 52% yield as a cluster of the dn-congeners with the isotopic composition d5 ∶ d6 ∶ d7 ∶ d8 ∶ d9 ∶ d10 = 4 ∶ 17 ∶ 33 ∶ 31 ∶ 12 ∶ 3. The average number of deuterium atoms (nD) incorporated was calculated as 7.3 D. Under the same conditions, the (24R)-24-methyl and -ethyl congeners of 3a were prepared from campesta-5,7-dien-3β-ol and stigmasta-5,7-dien-3β-ol, with 7.0 D and 6.7 D, respectively. Prolongation of the reaction time increased the level of incorporation due to the primary kinetic isotope effect of the deuterium atom. With a 48 h reaction, the composition ratio of 3a changed to d6 ∶ d7 ∶ d8 ∶ d9 ∶ d10 ∶ d11 ∶ d12 ∶ d13 = 2 ∶ 8 ∶ 22 ∶ 32 ∶ 24 ∶ 9 ∶ 2 ∶ 1, i.e., the average incorporation increased to 9.1 D. Analysis of the 1H NMR and MS spectra of 3a implicated all possible conjugated dienes across the B, C, and D rings in this migration reaction, at least in the 5α-series.